PROJECT SUMMARY: I propose a research program for this highly innovative award that aims to determine whether cell-free DNA (cfDNA) in cerebrospinal fluid and plasma can be used to identify neuronal- specific disease-associated genetic and epigenetic variation in patients with epilepsy. The candidate has expertise in the use of next generation DNA sequencing technology development and application to identify pathogenic variants that cause genetic disease. These skills will be applied to develop new experimental high-throughput and highly sensitive sequencing technologies to identify neuronal-specific alterations in cfDNA from patients with epilepsy. Epilepsy affects 1 in 26 individuals across the globe, including ~22 million people in the US alone. Since the introduction of high-throughput sequencing approaches, approximately 30-50% of patients with early-onset pediatric epilepsies have a molecular diagnosis, with the majority arising as a result of de novo pathogenic variation. Despite these progresses, the majority of patients remain without a diagnosis, despite strong evidence of a genetic etiology. It has been hypothesized that some of the remaining cases may be attributed to somatic mosaicism or epigenetic mutations. However, by and large these potentially disease-causing variations go undetected due to the inability to sample the affected tissue, the brain. We hypothesize that cfDNA in cerebrospinal fluid and even plasma may be a source for detecting these variants, as the cfDNA may originate from dying cells in the brain. We will characterize the origins of cfDNA in CSF and plasma in mice and humans using epigenomic profiling. Moreover, we aim to identify the genetic and epigenetic variations in genetic mouse models of epilepsy, as well as patients with epilepsy. This experimental model is highly transferable and may be applied to understand disease mechanisms and molecular diagnosis in other neurodevelopmental and neurological disorders, contributing more broadly to our understanding of disease and laying the foundation for the candidate?s new research program.